Spray -dried bacteriocin powder with anti-microbial activity

ABSTRACT

The production of a spray-dried bacteriocin lacticin 3147 powder is described. The powder is shown to have effective anti-microbial activity in a range of foodstuffs, namely infant milk formulations, powdered soup, yoghurt and cottage cheese. Increased anti-microbial activity was demonstrated when the lacticin 3147 powder was used in conjunction with increased hydrostatic pressure. The process comprises: inoculating a medium with a lacticin 3147-producing strain of bacteria, fermenting the inoculated medium, adjusting the pH of the fermentation to 6.3-6.7, inactivating the bacterial fermentate and evaporating the fermentate.

This application is a 371 of PCT/IE99/00058, filed Jun. 22, 1999.

The present invention relates to a spray-dried bacteriocin powder withanti-microbial activity, and to a method of producing the powder. Inparticular, the invention relates to a lacticin 3147 spray-dried powder.

1. Prior Art

The elimination of food spoilage and pathogenic organisms has become thefocus of much research since, in terms of individuals affected and thecost of treatment, food-borne illnesses have an enormous impact. It hasbeen estimated that microbial pathogens in food cause 6.5-33 millioncases of human illness annually in the U.S., at a cost of between$2.9-$6.7 billion dollars (2), with Gram-positive food-borne pathogensaccounting for between 25-55% of the costs. In recent years, consumerdemand for fresh minimally processed safe food, in addition to concernover the use of chemical preservatives in foods, has promptedsubstantial interest in the application of biopreservatives.Bacteriocins produced by lactic acid bacteria are seen as alternativesto traditional preservatives for ensuring food safety and potentialapplications in foods have been readily identified (21).

Nisin, a bacteriocin produced by certain strains of Lactococcus lactis,has been used successfully to control food spoilage, in a number ofdifferent foods, including cheeses, canned goods and dairy desserts(10). However, its use is subject to certain restrictions. It is mosteffective in foods with acidic pH (below pH 6.0) and low protein and fatcontent. It is poorly soluble above pH 6.0 and as such has limitedeffectiveness in many foods. A powdered form of nisin, Nisaplin (Aplinand Barrett, Towbridge, Wiltshire, U.K.) has been developed and is usedfor the preservation of foods.

In addition to the development of Nisaplin, other powderedbacteriocin-containing agents have been developed for the preservationof foods. Propionibacterium freundenreichii subsp. shermanni is used toproduce Microgard (Wesman Foods, Inc., Beaverton, Oreg.) bypasteurisation and drying of propionibacteria-fermented skim milk. It isestimated to be used in approximately one third of all cottage cheesemade in the US and is said to be inhibitory to most Gram-negativebacteria and some fungi (4). The active agents in Microgard includepropionic acid, acetic acid, diacetyl, lactic acid and a heat-stablepeptide of approximately 700 daltons which is considered to be the mostactive component.

Lacticin 3147 is a bacteriocin produced by L. lactis DPC3147 which has asimilar host range to that of nisin, in that it is inhibitory to a widerange of Gram-positive organisms, including Listeria, Clostridium spp.,Enterococcus, Staphylococcus and Streptococcus (17). Given that many ofthese organisms have been identified as agents of food spoilage andpathogenesis, the development of a lacticin 3147-based system forcontrol of these organisms has obvious attractions. This may be achievedin two ways. The first involves the use of starter cultures (includingtransconjugants) which produce lacticin 3147, and can be used in foodfermentations where these strains can be substituted for the originalstarter cultures. The genetic determinants for lacticin 3147 are encodedon a 60.2 kb plasmid, pMRC01 which has been fully sequenced (6) andwhich has been mobilised to a number of cheese starter cultures (3).Lacticin 3147 is the subject of PCT Application No. PCT/IE96/00022,published as WO 96/32482.

Recently, it has been shown that a lacticin 3147 producingtransconjugant can inhibit Listeria monocytogenes in Cottage cheese(13). This starter has also been used to control the proliferation ofnon-starter lactic acid bacteria in Cheddar cheese. The second approachto improving food safety through the use of lacticin 3147 involves thedevelopment of a spray-dried form of the bacteriocin. The advantage ofsuch a bio-active powder is that it could be applied as a foodingredient in a variety of foods. However, it is not at all apparentthat the bacteriocin is robust enough to withstand spray-drying andthere was the possibility that spray-drying would result in asignificant loss in bacteriocin activity.

OBJECT OF THE INVENTION

The object of the invention is to provide a lacticin 3147-enriched foodingredient for incorporation into foodstuffs. In particular, it is anobject to provide a spray-dried lacticin 3147 powder. It could not bepredicted that such a spray-dried powder could be produced sincespray-drying could have caused heat denaturation of the bacteriocin,bearing in mind that lacticin 3147 is composed of two peptides, both ofwhich are required for activity. Furthermore, dehydration couldirreversibly inactivate the bacteriocin.

Described herein is a whey based bio-active powder, with effectivenessin controlling two representative pathogens, L. monocytogenes andStaphylococcus aureus, in buffer at both neutral and acidic pH. Alsodescribed is its effectiveness in controlling L. monocytogenes in aninfant milk formulation and other foodstuffs. However, it will beapparent to those skilled in the art that the bacteriocin-powder of theinvention need not be dairy based and that it would also be possible toproduce a spray-dried bacteriocin based, for example, on other powders,synthetic materials or the like.

SUMMARY OF THE INVENTION

According to the present invention there is provided a process for theproduction of spray-dried lacticin 3147 powder comprising:

(a) inoculating a medium with a lacticin 3147-producing strain ofbacteria;

(b) fermenting the inoculated medium;

(c) adjusting the pH of the fermentation to pH 6.3 to 6.7;

(d) inactivating the bacterial fermentate;

(e) evaporating the fermentate of step (d).

The medium which may be inoculated with the bacteria can be selectedfrom milk or dairy-based powders including demineralised whey powder,reconstituted skimmed milk powder, whey protein concentrate powder,pasteurised milk, Cheddar cheese whey, or synthetic laboratory mediasuch as LM17 or TY broth or the like.

Preferably the inoculated medium is fermented at about 30° C. for about6 to 24 hours.

Preferably the pH of the fermentation is adjusted to about 6.5.

Suitably, the bacterial fermentate is inactivated by pasteurisation ortreating at ultra-high temperature.

Suitably, if the fermentate is pasteurised, it is pasteurised at about72° C. for about 15 seconds.

Preferably the inactivated fermentate is evaporated at about 60° C. toabout 40% total solids.

The concentrate of step (e) may then be cooled to about 32° C., seededwith lactose at about 0.1% w/w and allowed to crystallise at a coolingrate of about 1° C. per hour.

The crystallised concentrate is then spray-dried by methods known in theart.

The invention also provides a spray-dried lacticin 3147 powder which hasthe ability to inhibit organisms which are not resistant to lacticin3147 and which may suitably have an activity of about 40,240 au(arbitary units)/per ml.

The invention also provides a food product comprising a spray-driedlacticin 3147 powder as defined above. The food product may be an infantmilk formulation, a sauce, mayonnaise, a dessert, a yoghurt, a custard,a tinned food product such as a tinned vegetable or tinned meat product,a soup, a bakery product or similar products.

The food product may further have been subjected to increasedhydrostatic pressure during processing, suitably at a pressure of about150 to 800 MPa.

FIGURE LEGENDS

The present invention will now be described in greater detail withreference to the accompanying drawings in which:

FIG. 1. (A) Growth of L. lactis DPC3147 and lacticin 3147 production in10% reconstituted demineralized whey powder at 30° C., in pH controlledand uncontrolled conditions. (♦) cfu/ml with no pH control imposed, (▪)cfu/ml at constant pH of 6.0, () cfu/ml at constant pH of 6.5 and (O)cfu/ml at constant pH of 7.0. () AU/ml with no pH control, (□) AU/ml atconstant pH of 6.0, (▪) AU/ml at constant pH of 6.5 and () AU/ml atconstant pH of 7.0. (B) Inhibitory activity of lacticin 3147 against L.lactis HP when (a) grown with no pH control and when (b) grown at aconstant pH of 6.5.

FIG. 2. Schematic diagram of temperature profile and lacticin 3147activity during the manufacturing of lacticin 3147 powder.

FIG. 3. Effect of lacticin 3147 powder on the viability of Listeriamonocytogenes Scott A in buffer at 30° C. (A) at pH 5 and (B) at pH 7.(♦) no addition, (▪) addition of 10% lacticin 3147 powder.

FIG. 4. Effect of lacticin 3147 powder on the viability ofStaphylococcus aureus 10 in buffer at 30° C. (A) at pH 5 and (B) at pH7. (♦) no addition, (▪) addition of 15% lacticin 3147 powder.

FIG. 5. Effect of lacticin 3147 powder on the viability of L.monocytogenes Scott A when used as a component of infant milk formula.(▪) 15% lacticin powder, (▴) 10% lacticin powder, 5% infant milk powder,() 5% lacticin powder, 10% infant milk powder, (♦) 15% infant milkpowder.

FIG. 6. Effect of lacticin 3147 powder (10%) on the viability ofListeria monocytogenes Scott A in yoghurt. (▴) no lacticin 3147 added,(▪) 10% lacticin 3147 added. The 10% here refers to 10 g lacticin 3147powder added to 90 g yoghurt.

FIG. 7. Effect of lacticin 3147 powder (10%) on the viability ofListeria monocytogenes Scott A in cottage cheese. (♦) no lacticin 3147added, (▪) 10% lacticin 3147 added. The 10% here refers to 10 g lacticin3147 powder added to 90 g cottage cheese.

FIG. 8. Effect of lacticin 3147 powder (10%) on the viability ofBacillus cereus in (packet) soup.

(♦) no lacticin 3147 added,

(▪) 1% lacticin 3147 added,

(▴) 5% lacticin 3147 added,

() 10% lacticin 3147 added.

The 1, 5, 10% here refers to 1, 5 or 10 g lacticin 3147 powder added to99, 95 or 90 g packet soup powder, then reconstituted to themanufacturers instructions.

FIG. 9. Effect of lacticin 3147 powder (10%) on the viability ofListeria monocytogenes Scott A in (packet) soup.

(♦) no lacticin 3147 added,

(▪) 1% lacticin 3147 added,

(▴) 5% lacticin 3147 added,

() 10% lacticin 3147 added.

The 1, 5, 10% here refers to 1, 5 or 10 g lacticin 3147 powder added to99, 95 or 90 g packet soup powder, then reconstituted to themanufacturers instructions.

FIG. 10. The effect of increasing pressures on the activity of lacticin3147, (a) atmospheric pressure, (b) 200 MPa. (c) 400 MPa, (d) 600 MPaand (e) 800 MPa.

FIG. 11. The effect of high pressure and lacticin 3147 on L. innocuaDPC1770 viability.

EXAMPLE Material and Methods

Bacterial Strains and Culture Conditions

The bacteriocin producer L. lactis subsp lactis DPC3147 and thesensitive indicator strain L. lactis subsp lactis HP were routinelygrown at 30° C. in M17 (20; Oxoid Ltd., Basingstoke, Hampshire, England)supplemented with 0.5% (w/v) lactose. Other indicator strains usedincluded L. monocytogenes Scott A grown in Trypicase Soy Broth (TSB,Becton Dickinson and Co., Cockeysville, Md. 21030, USA) supplementedwith 0.6% (w/v) yeast extract (Oxoid), and Staphylococcus aureus 10 (DPCculture collection, Moorepark, Fermoy, Co. Cork, Ireland) grown in BrainHeart Infusion broth (BHI, Oxoid), both at 37° C. Solid media wasprepared by the addition of 1% bacteriological agar (Oxoid).

A number of different media were investigated for the production oflacticin 3147. These were made as 10% (w/v) solutions, apart frompasteurised whole milk and Cheddar cheese whey. The 10% solutions wereprepared from demineralized whey powder (95% demineralized),reconstituted skimmed milk powder (Dairygold, Mitchelstown, Co. Cork,Ireland) and whey protein concentrate powder (WPC35, 35% protein in drymatter, Moorepark Technology Ltd., Moorepark, Fermoy, Co. Cork,Ireland). The whey based solutions were sterilised by heating to 95° C.for 30 minutes. The skimmed milk powder solution was sterilised byautoclaving for 5 min at 121° C.

Bacteriocin Assay and Activity Determination

Bacteriocin activity was determined by the agar well diffusion assay asdescribed by Parente and Hill (15). Molten agar was seeded with anindicator strain and dispensed into petri dishes. Wells of approximately4.6 mm in diameter were bored in the agar and a 50 μl volume of a twofold serial dilution of a bacteriocin preparation was dispensed intoeach well. Bacteriocin solution was prepared by centrifuging the cultureand heat treating the supernatant at 70° C. for 10 minutes prior tocarrying out the dilution series. The plates were then incubated ateither 30° C. or 37° C., depending on the indicator strain used.Bacteriocin activity was calculated as the inverse of the last dilutionthat gave a definite zone of clearance after overnight incubation.Activity units (AU) were expressed per milliliter (1/dilution, ×20).

Controlled pH Fermentations.

Controlled pH fermentations were carried out over a 24 hour period, withslow agitation (approximately 20 rpm) at 30° C. A 1% inoculum of DPC3147was used to inoculate 100 ml of growth media. The pH of the growth mediawas kept at a constant value by the addition of 1.0 M NaOH on demand viaa 718 STAT Titrino (Metrohm, Ireland). Cell counts and bacteriocinactivity determinations were carried out at hourly if intervals for thefirst 10 hours, and a final sample was taken after 24 hours.

Production of a Spray Dried Lacticin 3147 Powder.

A 170 L volume of demineralized whey powder (10% total solids) wasinoculated with 1% DPC3147 and the pH of the 24 hour fermentation wascontrolled by the addition of 2.5M NaOH on demand (pH 6.5). Thefermentate was then pasteurised at 72° C. for 15 sec using an APV SSPpasteurizer (APV, Silkborg, Denmark). The pasteurised fermentate wasthen evaporated at 60° C. to 40% total solids using a single effectfalling film evaporator (Anhydro model F1 Lab). The resultingconcentrate was cooled to 32° C., seeded with lactose (0.1% w/w) andallowed to pre-crystallize overnight at a cooling rate of 1° C. perhour. The pre-crystallized concentrate was then spray-dried using nozzleatomization in an Anhydro spray drier (Anhydro model Lab 3) at an airinlet temperature of 190° C. and a 90° C. outlet temperature. The powderwas aliquoted, sachet packed in foil-lined sample bags and stored at 4°C. Bacteriocin activity was assessed at each step during the process.

Effect of Lacticin 3147 Powder Against Pathogens in Buffer

Sensitive cells were grown to mid-exponential phase, washed andresuspended at approximately 10⁷-10⁸ cfu/ml in 2.5 mM sodium phosphatebuffer, pH 7.0 or pH 5.0, and 2.5 mM sodium phosphate buffer, pH 7.0 orpH 5.0 supplemented with 10 mM glucose. Lacticin 3147 powder was added(at different concentrations depending on the sensitive strain underinvestigation) and samples were taken at appropriate time intervals overa 3 hour period to determine the viable cell count.

Effect of Lacticin 3147 Powder Against L. monocytogenes in an InfantMilk Formulation

Lacticin 3147 powder was added to a commercially available infant milkformula, [ingredients listed as follows: demineralized whey powder,vegetable oils, lactose, skimmed milk, calcium carbonate, potassiumcitrate, calcium chloride, sodium citrate, magnesium chloride, vitaminC, emulsifier (soya lecithin), taurine, potassium hydroxide, ironsulphate, zinc sulphate, vitamin E, nicotinamide, pantothenic acid,vitamin A, copper sulphate, citric acid, thiamin, vitamin B₆, (carotene,manganese sulphate, potassium iodide, folic acid, vitamin K, sodiumselenite, vitamin D, biotin). Manufacturers instructions indicate thatthe final liquid for infant consumption is a 15% solution (w/v). Inexperiments the 15% (w/v) infant milk powder was replaced with either 5%(w/v) lacticin powder and 10% (w/v) infant milk powder, or with 10%(w/v) lacticin powder and 5% (w/v) infant milk powder. L. monocytogenescells were grown to mid-exponential phase, washed and resuspended atapproximately 10⁴ cfu/ml in the various infant milk formulations at 30°C. and samples were taken at appropriate time intervals over a 3 hourperiod to determine the viable cell count.

Preparation of Lacticin 3147 for Use in High Pressure InactivationStudies

For the inactivation of Staph. aureus ATCC6538 a liquid preparation oflacticin 3147 was prepared using hydrophobic adsorption chromatography.For studies on inactivation of L. innocua DPC1770 a food grade powderedpreparation of lacticin 3147 was manufactured as described above withthe following modification; a 1% demineralised whey powder solution wasfermented with L. lactis subsp. lactis DPC3147 under pH controlledconditions of pH 6.0 for 18 hours.

Activity of both lacticin 3147 preparations was determined by the agarwell diffusion assay as described by Parente and Hill (15). Molten agarwas seeded with the indicator strain L. lactis subsp. lactis HP anddispensed into petri dishes. Wells of approximately 6.0 mm in diameterwere bored in the agar and a 50 μl volume of a two fold serial dilutionof a bacteriocin preparation was dispensed into each well. The plateswere then incubated at 30° C. Bacteriocin activity was calculated as theinverse of the last dilution that gave a definite zone of clearanceafter overnight incubation. Activity units (AU) were expressed permilliliter (1/dilution, ×20). Activity may also be expressed as zonediameter (mm), where the diameter of the first zone (neat, undilutedsample) of the dilution series is recorded.

Effect of High Pressure on Staph. aureus ATCC6538 and L. innocua DPC1770Viability

Staph. aureus ATCC6538 cells were resuspended in 10% RSM and aliquotedinto sterile 700 μl PCR eppendorfs prior to placing in sterile stomacherbags (Seward Ltd., London, UK). Ten millilitre volumes of L. innocuaDPC1770 cells were resuspended in 20% reconstituted demineralised wheypowder aliquoted into sterile stomacher bags. Samples were individuallyvacuum sealed prior to placing in the pressure vessel (Stansted FluidPower Ltd., Stansted, England). The vessel consisted of astainless-steel cylinder (37 mm diameter×300 mm height) filled with a15% (v/v) caster oil in ethanol solution which acts as the hydrostaticpressurisation medium. Samples were treated for 30 min at 25° C. in thepressure range 150 to 600 MPa, in addition to a control sample beingheld at atmospheric pressure (0.1 MPa). All experiments were carried outin duplicate. The chamber temperature was determined by means of athermoregulating system which circulated to maintain the chambertemperature.

Effect of High Pressure on Lacticin 3147 Activity

To determine the effect of high pressure on lacticin 3147 activity,reconstituted lacticin 3147 powder and aliquots of liquid lacticin 3147were vacuum sealed and exposed to pressures ranging from 100 to 800 Mpaas described above. Pressurised and non-pressurised solutions oflacticin 3147 were heat treated at 80° C. for 10 minutes prior tocarrying out activity determination by the well diffusion assay using L.lactis HP as an indicator strain.

Results

The objective of this research was to develop a powdered form oflacticin 3147 suitable for use as an ingredient which could help in thecontrol of undesirable micro-organisms in foods. Following theoptimization of lacticin 3147 production a scale-up fermentation wascarried out and the fermentate was spray-dried to form abacteriocin-rich powder. This powder was assessed in both a buffer andan infant milk food system for it, ability to inhibit pathogens.

Lacticin 3147 Production in Various Media

Following inoculation of DPC3147 (1%) and overnight incubation at 30°C., lacticin 3147 activity was assessed in a number of different growthmedia. Most of the media were dairy based, but two synthetic media werealso included (LM17 and TY). Results of production of lacticin 3147 (seeTable 1) demonstrated that activity was high in almost all the dairybased media (1,280 to 2,560 AU/ml) apart from WPC35 (320 AU/ml). Highestlevels of lacticin 3147 activity were found in Cheddar cheese whey,whole milk and LM17 (2,560 AU/ml). Both 10% reconstituted demineralizedwhey powder and 10% reconstituted skimmed milk powder gave activity of1,280 AU/ml. Lower levels of lacticin 3147 activity were observed in TYbroth (640 AU/ml).

Since demineralized whey powder is a commercially and readily available,and good lacticin 3147 activity was observed in this media, furtherinvestigations into the optimization of lacticin 3147 production indemineralized whey powder was carried out.

Optimization of Lacticin 3147 Production in 10% ReconstitutedDemineralized Whey Powder

Bacteriocin production and viable cell counts in pH-controlled andpH-uncontrolled fermentations revealed that increased levels of lacticin3147 could be produced by maintaining the pH of the growth mediaconstant, at pH 6.5 (FIG. 1). Levels of bacteriocin activity reached10,240 AU/ml in 10% reconstituted demineralized whey powder when the pHof the growth media was held constant at pH 6.5 (FIG. 1B(a)) compared to640 AU/ml when no pH control was imposed (FIG. 1B(b)). At both pH 6.0and pH 7.0 lacticin activity reached 5120 AU/ml. Results of viable cellcounts over a 24 hour period indicated that increased bacteriocinactivity corresponded to higher cell densities. Without pH controlviable cell counts reached 1×10⁹ cfu/ml, whereas when the pH of thegrowth media was maintained at a constant pH of 6.5 viable cell countsreached 3.8×10⁹ cfu/ml (FIG. 1A). With pH control at 6.0 and 7.0 viablecell counts reached 2.5×10⁹ cfu/ml.

Production of Lacticin 3147 Powder

A spray-dried lacticin 3147 preparation was manufactured as described inmaterials and methods. During the manufacturing process bacteriocinactivity was assessed at each step, using L. lactis HP as the indicatorstrain (FIG. 2). Following the pH controlled fermentation (in 10%reconstituted demineralized whey powder) bacteriocin activity was 10,240AU/ml. The fermentate was subjected to pasteurisation to inactivate thebacteriocin producing culture DPC3147. Pasteurisation had no effect onbacteriocin activity (FIG. 2). Evaporation (from 10% total solids to 40%total solids) led to a concentration of the fermentate and resulted inan increase in bacteriocin activity to 40,960 AU/ml. Following overnightcrystallisation, the activity of the concentrate remained stable. Spraydrying of the concentrate resulted in the production of an activepowder. When resuspended at a concentration of 50 mg/ml (5% solids) thespray dried powder contained 5,120 AU indicating that the activity ofthe lacticin powder was 102,400 AU/g (100% solids). Lacticin 3147activity expressed as AU/g of dry matter remained constant throughoutmanufacture at 102,400 AU/g, indicating that no loss in bacteriocinactivity occurred during processing.

The inhibitory activity of the bacteriocin-enriched powder wasattributed to the action of lacticin 3147 rather than other fermentationmetabolites such as lactic acid, since it inhibited a sensitive L.lactis MG1614, but did not show any inhibitory effect against atransconjugant containing the pMRC01 plasmid.

Effect of Lacticin 3147 Powder on Pathogens

The lacticin 3147 enriched demineralized whey powder (lacticin 3147powder) was investigated for its ability to inhibit two food-bornepathogens. The inhibitory effect of the powder was investigated at pH 5and at pH 7, in the presence and absence of 10 mM glucose. Theeffectiveness of a 10% (w/v) solution of lacticin 3147 powder againstmid-exponential growth phase cells of L. monocytogenes Scott Ademonstrated that approximately a 3.3 log kill (99.95% kill) could beachieved at pH 5 within 3 hours at 30° C. (FIG. 3A). Killing of L.monocytogenes Scott A with a 10% (w/v) solution of lacticin powder wasslightly more effective at pH 7 (FIG. 3B). A 3.8 log kill (99.98% kill)was observed within 3 hours at 30° C.

S. aureus 10 was found to be more resistant than L. monocytogenes ScottA to the action of the lacticin enriched powder, for this reason a 15%solution of the powder was used. The effectiveness of a 15% (w/v)solution of lacticin 3147 powder against mid-exponential phase cells ofS. aureus 10 resulted in approximately a 1.1 log kill (90.4% kill) at pH5 within 3 hours at 30° C. (FIG. 4A). The killing effect of a 15%solution (w/v) of lacticin powder increased dramatically at pH 7, wherealmost a 4 log kill (99.98% kill) of S. aureus 10 was observed within 3hours at 30° C. (FIG. 4B). The inclusion of 10 mM glucose resulted onlyslight increases in the level of cell deaths for either L. monocytogenesScott A or S. aureus 10 (results not shown).

Effect of Lacticin 3147 Powder Against L. monocytogenes Scott A in anInfant Milk Formulation

To evaluate the effectiveness of the lacticin 3147 powder in a foodsystem experiments were carried out in an infant milk formula, sincethis is an example of a food destined for a high-risk consumer whichcontains demineralized whey powder as a major constituent. Resultsindicated greater that a 99% kill of L. monocytogenes Scott A resultedwhen part of the infant milk formulation was substituted with either twothirds (10% lacticin powder and 5% infant milk powder) or one thirdlacticin 3147 powder (5% lacticin powder and 10% infant milk powder)(FIG. 5). Counts here were reduced from approximately 7×10⁴ cfu/ml to3×10¹ cfu/ml within 3 hours at 30° C. In the control culture with nolacticin 3147 powder present counts increased from approximately 10⁴cfu/ml to approximately 10⁵ cfu/ml within the same time period.

Application of Lacticin 3147 Powder in a Range of Foods

Powdered lacticin 3147 has been assessed for the inhibition of foodspoilage and pathogenic micro-organisms in a number of food systemsincluding infant food formula, powdered soup, cottage cheese and naturalyoghurt. The following are specific examples of the use of lacticin 3147to inhibit pathogens in food systems.

The ability of the lacticin 3147 powder to inhibit Listeriamonocytogenes Scott A was initially investigated in an infant milkformulation as described above. To further investigate the inhibitoryeffect of the lacticin 3147 powder, inactivation trials were carried outagainst a number of different micro-organisms in natural yoghurt,cottage cheese and reconstituted powdered soup, with pHs of 4.5, 4.4 and6.6 respectively.

The effect of 10% lacticin 3147 powder on the inhibition of Listeriamonocytogenes Scott A (10⁴ cfu/ml) in natural yoghurt demonstrated thatgreater than 98.3% of the culture was killed within 5 minutes at 30° C.Within 60 minutes no viable cells remained, (FIG. 6).

In the case of cottage cheese inoculated with 10⁴ cfu/ml Listeriamonocytogenes 40% of the population was killed within 5 minutes at 30°C. in the presence of a 10% lacticin 3147 powder. After 160 minutes only14% of the population remained viable, (FIG. 7).

The effect of 1, 5 and 10% concentrations of lacticin 3147 in powderedsoup against Bacillus cereus at 30° C., demonstrated that following 24hours incubation greater than a 99.9% kill was observed in the presenceof the 5 and 10% lacticin 3147 powder concentrations. In the case of the1% lacticin 3147 concentration 17% of the population survived, (FIG. 8).

A similar study was carried out to determine the effect of 1, 5 and 10%concentrations of lacticin 3147 powder on the survival of Listeriamonocytogenes Scott A in powdered soup. A 1% concentration of lacticinwas ineffective at inhibiting Scott A within 24 hours, whereas at a 5%concentration greater than 10% of the population were inhibited. At aconcentration of 10% greater than 40% of the culture was inhibited,(FIG. 9).

From these results it can be seen that a powdered form of lacticin 3147has indeed many applications in food safety for the control of foodpathogens and spoilage organisms.

Effect of Hydrostatic Pressure

The use of hydrostatic pressure and lacticin 3147 treatments wereevaluated in milk and whey with a view to combining both treatments forimproving the quality of minimally processed dairy foods. The system wasevaluated using two foodborne pathogens, Staphylococcus aureus ATCC6538and Listeria innocua DPC1770. Trials against Staph. aureus ATCC6538 wereperformed using concentrated lacticin 3147 prepared from culturesupernatant. Results demonstrated greater than an additive effect whenboth treatments were used in combination, for example, the combinationof 250 MPa (2.2 log reduction) and lacticin 3147 (1 log reduction)resulted in more than 6 logs of kill (FIG. 10). Similar results wereobtained when a foodgrade powdered form of lacticin 3147 (developed froma spray dried fermentation of reconstituted demineralised whey powder)was evaluated for the inactivation of L. innocua DPC1770 (FIG. 11).Furthermore, it was observed that treatment of lacticin 3147preparations with pressures greater than 400 MPa yielded an increase inbacteriocin activity (equivalent to a doubling of activity). Theseresults indicate that a combination of high pressure and lacticin 3147may be suitable for improving the quality of minimally processed foodsat lower hydrostatic pressure levels.

Discussion

The development of a whey based bio-active food ingredient was achievedfollowing investigations into lacticin 3147 production in differentmedia. Lacticin 3147 activity was high in all of the dairy based mediainvestigated, apart from whey protein concentrate (WPC35). A possibleexplanation for the low level of activity in the whey proteinconcentrate could be that bacteriocin activity fractionated into thepellet upon centrifugation, prior to assaying for activity. Twosynthetic media were investigated for lacticin 3147 production, LM17broth (20) and TY broth (15). Levels of lacticin 3147 activity in LM17were comparable to dairy based media, but this is not unexpected, sincethis media was developed for the cultivation of lactococci. However, TYbroth, in which low levels of lacticin 3147 activity was observed, wasdeveloped to yield optimal bacteriocin (enterocin 1146) production whileminimising peptide levels in the medium (to eliminate peptides that mayinterfere with purification). For the development of a powder the use ofthe most cost effective growth media is obviously advantageous.Demineralized whey powder, a readily available and cost effective medium($20 per 25 Kg) was investigated for the optimization of lacticin 3147production. However, other suitable growth media could be used, asdescribed above.

The effect of pH on bacteriocin production has been well documented, andfor a number of bacteriocin-producing strains control of pH duringgrowth results in higher bacteriocin titres (11, 14, 18). Lacticin 3147activity increased dramatically when the pH of the growth media was heldconstant at pH 6.5, 5 highest bacteriocin titres and highest cellnumbers were observed at this pH. Lowest bacteriocin titres and lowestcell numbers were observed when no pH control was imposed. Increasedbacteriocin activity corresponded with increased cell numbers.

Once lacticin 3147 production had been optimized in 10% reconstituteddemineralized whey powder a large-scale fermentation was set up togenerate enough fermentate for spray drying. The production of an activespray dried powder demonstrated the resilience of the bacteriocin, tothe extremes of the processing conditions. Activity was detectedthroughout the process and the final powder had an activity of 102,400AU/g dry matter, equivalent to the activity present at the beginning ofthe process. This unexpected result is significant in that it suggeststhat no loss in activity occurred during production.

Assessment of the inhibitory activity of the bio-active powderdemonstrated that it is capable of inhibiting both L. monocytogenes andS. aureus at pH 5 and at pH 7. In both cases the bio-active powderexhibited enhanced killing ability at neutral pH. This is a significantfinding, since Nisaplin, a fermented food ingredient for extension ofproduct shelf life and prevention of spoilage is known to be mosteffective at acidic pH (below pH 6.0). The development of a foodingredient capable of killing Gram-positive bacteria at neutral pHindicates that the lacticin 3147 powder may be suitable forincorporation into a wide range of foods, that hitherto had noopportunity for the prevention of food spoilage/pathogenesis apart fromthe inclusion of chemical preservatives.

The mechanism of action of lacticin 3147 has been elucidated (12). Itinduces cell death by permeabilising the membranes of sensitive cellsthrough pore formation, allowing the efflux of K⁺ ions and phosphate.This action results in the dissipation of the proton motive force,hydrolysis of intracellular ATP and ultimately leads to cell death.Energised cells are more susceptible to the action of lacticin 3147.Cells incubated in the presence of lacticin powder combined with 10 mMglucose demonstrated slight increases in killing efficiency (apart fromS. aureus 10 at pH 7, results not shown). This is in keeping withresults reported by McAuliffe et al., (12), where energised cells wereobserved to be more sensitive to lacticin 3147. Energised cells have aproton motive force which may favour the insertion of lacticin 3147molecules into the membrane, as is the case with nisin, a lantibioticpore former (7, 8).

The development of a powdered form of lacticin 3147 would allow it to beapplied to a number of food systems. Since the existing lacticin 3147powder has been developed from a demineralized whey powder, this powderhas applications in all foods where demineralized whey powder is anexisting ingredient. For example demineralized whey powder isincorporated into a number of foods including infant milk formulations.Results presented in this paper demonstrate the ability of this powderto effectively inactivate 99% of L. monocytogenes Scott A spiked intoinfant formula, where part of the infant milk powder had beensubstituted with the lacticin 3147 powder. Infant milk formulations aremanufactured to the highest of standards and incidents of food-borneillness associated with such foods are rare. However more than manyother foods infant milk formulas are susceptible to contaminationthrough domestic contamination, putting the health of infants at risk.For this reason the inclusion of a lacticin 3147 enriched powder in suchformulations may offer increased protection in the event ofcontamination, which would be beneficial to both producers andconsumers.

For manufacturers already using demineralized whey powder as a foodingredient it should prove possible to substitute this powder (eitherpartially or fully) with a bio-active demineralized whey powder tofurther safe guard food products from spoilage and pathogenicGram-positive organisms. And indeed, for manufacturers who do not usedemineralized whey powder as a food ingredient the inclusion of lowlevels of the bio-active powder could be sufficient to confer enhancedprotection without affecting the sensory or functional characteristicsof these foods. It is, however, also apparent that a spray-driedlacticin 3147 powder based on a medium other than whey powder, would beobtainable by this invention. Such a powder has potential forapplication as a substitute in areas where whey powder is not utilised,with the same beneficial effects.

SUMMARY

The broad-spectrum bacteriocin lacticin 3147, produced by Lactococcuslactis DPC3147, is inhibitory to a wide range of Gram-positive foodspoilage and pathogenic organisms. A 10% solution of demineralized wheypowder was fermented with DPC3147 at a constant pH of 6.5. Thefermentate was spray dried and the resulting powder exhibited inhibitoryactivity. The ability of the lacticin 3147-enriched powder to inhibitListeria monocytogenes Scott A and Staphylococcus aureus 10 was assessedin buffer at both acidic (pH 5) and neutral pH (pH 7). In addition, theability of the powder to inhibit L. monocytogenes Scott A in an infantmilk formulation was assessed. Resuspension of 8.3 log mid-exponentialphase L. monocytogenes Scott A cells in a 10% solution of the lacticin3147-enriched powder resulted in a 1000 fold reduction in viable cellsat pH 5 and pH 7, after 3 hours at 30° C. In the case of S. aureus 10,resuspension of 2.5×10⁷ mid-exponential phase cells in a 15% solution ofthe lacticin 3147-enriched powder at pH 5 resulted in only a 10 foldreduction in viable cell counts, compared to a 1000 fold reduction at pH7, following incubation for 3 hours at 30° C. In an infant milkformulation the use of the lacticin 3147 powder resulted in greater thana 99% kill of L. monocytogenes within 3 hours at 30° C. Similarily, thelacticin 3147 powder was shown to be effective in inhibiting foodspoilage in powdered soup, yoghurt and cottage cheese. Furthermore, thecombination of hydrostatic pressure and lacticin 3147 causes increasedkilling making this an attractive method of preventing spoilage inminimally processed foodstuffs. Thus this bio-active lacticin 3147 foodingredient will find applications in many different foods, includingthose with pH close to neutrality.

References

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3. Coakley, M., G. F. Fitzgerald and R. P. Ross. 1997. Application andevaluation of the phage resistance and bacteriocin-encoding plasmidpMRC01 for the improvement of dairy starter cultures. Appl. Environ.Microbiol. 63:1434-1440

4. Daeschel, M. A. 1989. Antimicrobial substances from lactic acidbacteria for use as food preservatives. Food Technol. 43:164-167

6. Dougherty, B., C. Hill, J. F. Weidman, D. R. Richardson, J. C. Venterand R. P. Ross. Sequence and analysis of the 60 kb conjugative,bacteriocin producing plasmid pMRC01 from Lactococcus lactis DPC3147.Mol. Microbiol. (in press).

7. Driessen, A. J. M., H. W. van der Hooven, W. Kuiper, M. van de Kamp,H.-G. Sahl, R. N. H. Konings, and W. N. Konings. 1995. Mechanisticstudies of lantibiotic-induced permeabilisation of phospholipidvesicles. Biochem. 34:1606-1614.

8. Garcia-Garcera, M. J., M. G. M. Elferink, A. J. M. Driessen, and W.N. Konings. 1993. In vitro pore-forming activity of the lantibioticnisin: role of protonmotive-force and lipid composition. Eur. J.Biochem. 212:417-422.

10. Hurst, A. 1983. Nisin and other inhibitory substances from lacticacid bacteria, p. 327-351. In P. M. Davidson and A. L. Branen, (ed.),Antimicrobials in food, Marcel Dekker, New York.

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12. McAuliffe, O., M. P. Ryan, R. P. Ross, C. Hill, P. Breeuwer and T.Abee. 1998. Lacticin 3147: a broad spectrum bacteriocin whichselectively dissipates the membrane potential. Appl. Environ. Microbiol.64:439-445.

13. McAuliffe, O., C. Hill and R. P Ross. 1998. Inhibition of Listeriamonocytogenes in Cottage cheese manufactured with a lacticin 3147producing starter culture. Submitted for publication: J. Appl.Microbiol.

14. Muriana, P. M., and T. R. Klaenhammer. 1987. Conjugal transfer ofplasmid encoded determinants for bacteriocin production and immunity inLactobacillus acidophilus 88. Appl. Environ. Microbiol. 53:553-560.

15. Parente, E., and C. Hill. 1992. A comparison of factors affectingthe production of two bacteriocins from lactic acid bacteria. J. Appl.Bacteriol. 73:290-298.

17. Ryan, M. P., M. C. Rea, C. Hill and R. P. Ross. 1996. An applicationin Cheddar cheese manufacture for a strain of Lactococcus lactisproducing a novel broad-spectrum bacteriocin, lacticin 3147. Appl.Environ. Microbiol. 62:612-619.

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20. Terzaghi, B. E., and W. E. Sandine. 1975. Improved medium for lacticstreptococci and their bacteriophages. Appl. Environ. Microbiol.29:807-813.

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The words “comprises/comprising” and the words “having/including” whenused herein with reference to the present invention are used to specifythe presence of stated features, integers, steps or components but doesnot preclude the presence or addition of one or more other features,integers, steps, components or groups thereof.

TABLE 1 Lacticin 3147 activity in various media after overnightincubation at 30° C. Lacticin 3147 activity Growth Media (AU/ml) Cheddarcheese whey 2560 Whole milk 2560 Reconstituted skimmed milk powder 1280Reconstituted demineralised whey powder 1280 Whey protein concentrate(WPC3S) 320 LM17 2560 TY broth 640

What is claimed is:
 1. A process for the production of a spray-driedconcentrate comprising lacticin 3147, for use as a food ingredient,comprising: (a) inoculating a milk or dairy based medium with a lacticin3147-producing strain of bacteria; (b) fermenting the inoculated medium;(c) adjusting the pH of the fermentation to a pH ranging from 6.3 to6.7; (d) inactivating the bacteria within the fermentate; and (e)evaporating the fermentate of step (d) thereby producing the lacticin3147 concentrate for use as a food ingredient.
 2. A process as claimedin claim 1, wherein the medium of step (a) is selected from the groupconsisting of milk, reconstituted dairy-based powders, reconstituteddemineralized whey powder, reconstituted skimmed milk powder,reconstituted whey protein concentrate powder, pasteurized whole milk,Cheddar cheese whey, reconstituted yeast powders, and syntheticlaboratory-type media.
 3. A process as claimed in claim 1 or 2, whereinthe evaporation step of step (e) comprises cooling the fermentate ofstep (d), seeding it with lactose at about 0.1% w/w and crystallizing ata cooling rate of about 1° C. per hour.
 4. A process as in claim 1,wherein the inoculated medium of step (b) is fermented at about 30° C.for about 6 to 24 hours.
 5. A process as in claim 1, wherein the pH ofthe fermentation is adjusted in step (c) to about pH 6.5.
 6. A processas in claim 1, wherein the fermentate of step (d) is inactivated bypasteurization or ultra-high temperature treatment.
 7. A process asclaimed in claim 6, wherein said pasteurization step comprises heatingat about 72° C. for about 15 minutes.
 8. A process as in claim 1,wherein step (e) comprises evaporating said bacteria fermentate at about60° C. to about 40% total solids.
 9. A process as in claim 1, furthercomprising the step of spray-drying the concentrate.
 10. A concentratecomprising a food-grade spray-dried lacticin 3147 produced by theprocess of any one of claims 1 to
 9. 11. A spray-dried food-grade powdercontaining lacticin 3147 having the ability to inhibit organisms whichare not resistant to lacticin 3147, and having an activity of greaterthan about 20,000 AU/ml.
 12. A food product comprising a lacticin 3147enriched spray-dried food-grade fermentate produced by the process ofany one of claims 1 to 9 and a foodstuff.
 13. The food product asclaimed in claim 12, wherein said product is selected from the groupconsisting of an infant milk formulation, a sauce, a mayonnaise, adessert including a custard, a tinned food, a yogurt, a soup and abakery product.
 14. A food product as claimed in claim 12 or 13, whichhas been subjected to hydrostatic pressure in the range from about 150MPa to about 800 MPa.